PROJECT SUMMARY/ABSTRACT Tumor-targeting bacteria offer a number of advantages over other cancer drug delivery systems, including targeting of multiple tumors from a distant inoculation site, selective intratumoral replication, a high degree of attenuation and safety, and the ability to express anti-cancer proteins directly within the tumor. Remarkably, attenuated Salmonella localize within solid tumors at levels at least 1000 times greater than other tissues. Human clinical studies have validated the safety of intravenously administered attenuated Salmonella mutant VNP20009, established tolerated multiple doses, and have shown that tumor targeting occurs in some patients. However, no anti-tumor activity was observed, even in patients in whom the Salmonella were verified to have colonized their tumors. We hypothesize that the lack of antitumor efficacy in the human clinical trials can be overcome by engineering antitumor apoptosis (programmed cell death) -inducing cytotoxic proteins that are able to selectively kill tumor cells, and that the ability of these proteins to kill cancer cells can be enhanced by blocking their degradation by tumor proteases through co-expression of protease inhibitors. The specific aims are designed to test the tumor-selective toxin generated during the initial SC3 funding period in murine models of cancer. The specific aims are also directed toward generation and analysis of individual protease inhibitor combinations expressed by VNP20009 in order to prevent proteolytic degradation of the therapeutic protein and develop tumor-targeted Salmonella vectors with enhanced antitumor activity. VNP20009 expressing a tumor-cell targeted toxin will be analyzed alone and in combination with one or more protease inhibitors using optical imaging of the bacterial interaction with murine tumor models of highly aggressive breast cancer. The results are expected to indicate the effectiveness of the targeted toxin and the effect(s) of the protease inhibitors alone and in combination with the toxin. These results have the potential to improve VNP20009 without increasing its toxicity, and therefore could lead to translational studies in humans. .